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COTI-2 reactivates mutant p53 and inhibits growth of triple-negative breast cancer cells

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Abstract

Purpose

Triple-negative breast cancer (TNBC) currently lacks an approved targeted therapy. The tumour suppressor TP53 gene is mutated in approximately 80% of TNBC cases. COTI-2 is a third-generation thiosemicarbazone engineered for high efficacy and low toxicity which acts by reactivating mutant p53 to a WT form. The aim of this study was to investigate COTI-2 as a targeted therapy for TNBC patients.

Methods

Using a panel of 18 breast cell lines, we carried out MTT assay. p53 protein folding was determined by immunofluorescent staining with the p53 mutant-specific antibody PAb240 and the p53 WT-specific PAb1620. Surface plasmon resonance was used to determine binding affinity of COTI-2 to full length (FL) p53, and the DNA-binding domain (DBD). Flow cytometry was used to measure apoptosis.

Results

TNBC cell lines were significantly more responsive to COTI-2 than non-TNBC cell lines (p = 0.04). Furthermore, lower IC50 values were found in p53 mutant compared to p53 WT cells (p = 0.001). COTI-2 was shown to bind to FL and DBD of mutant p53. Treatment resulted in an increase in staining with PAb1620 which coincided with a decrease in staining with PAb240, suggesting refolding of the mutant protein. In addition, COTI-2 was found to induce apoptosis in TNBC cell lines.

Conclusion

We conclude that targeting mutant p53 with COTI-2 is a potential approach for treating p53-mutated TNBC.

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Data availability

The p53 mutational status of the cell lines used in this study was sourced from the COSMIC database, available at https://cancer.sanger.ac.uk/cell_lines.

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Acknowledgements

We thank the BREAST-PREDICT (CCRC13GAL) program of the Irish Cancer Society and the Cancer Clinical Research Trust for funding this work. We also thank Professor Alan Fersht (MRC Laboratory of Molecular Biology, Cambridge) for supplying the full length and the mutated p53-R175H DNA-binding domain of p53.

Funding

This study was funded by the Irish Cancer Society as part of the BREAST-PREDICT Collaborative Cancer Research Centre (CCRC13GAL) and the Cancer Clinical Research Trust.

Author information

Authors and Affiliations

  1. UCD School of Medicine, Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Dublin 4, Ireland

    Naoise C. Synnott & Michael J. Duffy

  2. UCD School of Biomolecular & Biomedical Science, University College Dublin, Dublin 4, Ireland

    David O’Connell

  3. Department of Medical Oncology, St. Vincent’s University Hospital, Dublin 4, Ireland

    John Crown

  4. UCD Clinical Research Centre, St. Vincent’s University Hospital, Dublin 4, Ireland

    Michael J. Duffy

Authors
  1. Naoise C. Synnott
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  2. David O’Connell
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  4. Michael J. Duffy
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Contributions

MJD conceived the original idea. MJD and JC supervised the project. DOC designed, carried out and analysed the surface plasmon resonance experiments. NCS carried out all other experiments and data analysis. All authors contributed to results interpretation. NCS and MJD wrote the manuscript with input from DOC.

Corresponding author

Correspondence to Michael J. Duffy.

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Conflict of interest

NCS, DOC and MJD have no conflict of interest to report. JC reports the following: Research Funding: Puma, GSK, Roche, BI Honoraria: Eisai, Merck Serono, Pfizer, BI, Puma, Seattle Genetics, Genomic Health, Travel grants: AbbVie, Pfizer, MSD, Stock/Ownership: Oncomark.

Ethical approval and consent to participate

No ethical approval was required for this study, due to the fact that purchased cell lines were used.

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All authors give consent to publish this data. No human tissue was used in this study.

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Synnott, N.C., O’Connell, D., Crown, J. et al. COTI-2 reactivates mutant p53 and inhibits growth of triple-negative breast cancer cells. Breast Cancer Res Treat 179, 47–56 (2020). https://doi.org/10.1007/s10549-019-05435-1

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  • DOI: https://doi.org/10.1007/s10549-019-05435-1

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